Composition and method to alleviate joint pain

ABSTRACT

Beneficial and synergistic effects for alleviating joint pain and symptoms of osteoarthritis and/or rheumatoid arthritis have been found with krill oil and/or marine oil in combination with other active constituents, including astaxanthin and polymeric hyaluronic acid or sodium hyaluronate (hyaluronan) in an oral dosage form.

RELATED APPLICATION(S)

This application is a divisional application of Ser. No. 12/840,372filed Jul. 21, 2010, which is based upon prior filed provisionalapplication Ser. No. 61/227,872, filed Jul. 23, 2009; and provisionalapplication Ser. No. 61/345,652, filed May 18, 2010, the disclosures ofwhich are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to treating and alleviating symptoms ofosteoarthritis and/or rheumatoid arthritis using therapeuticcompositions and methods derived from krill extracts and/or marine oilcompositions and synergistic additives.

BACKGROUND OF THE INVENTION

The use of krill and/or marine oil are disclosed in U.S. PatentPublication Nos. 2004/0234587; 2004/0241249; and 2007/0098808, thedisclosures which are hereby incorporated by reference in theirentirety. The beneficial aspects of using krill and/or marine oil areshown also in a research paper published by L. Deutsch as “Evaluation ofthe Effect of Neptune Krill Oil on Chronic Inflammation and ArthriticSymptoms,” published in the Journal of the American College ofNutrition, Volume 26, No. 1, 39-49 (2007), the disclosure which ishereby incorporated by reference in its entirety.

The published '587, '249 and '808 applications discuss the beneficialaspects of using krill oil in association with pharmaceuticallyacceptable carriers. As an example, this krill and/or marine oil can beobtained by the combination of detailed steps as taught in the '808application, by placing krill and/or marine material in a ketonesolvent, separating the liquid and solid contents, recovering a firstlipid rich fraction from the liquid contents by evaporation, placing thesolid contents and organic solvent in an organic solvent of the type astaught in the specification, separating the liquid and solid contents,recovering a second lipid rich fraction by evaporation of the solventfrom the liquid contents and recovering the solid contents. Theresultant krill oil extract has also been used in an attempt to decreaselipid profiles in patients with hyperlipidemia. The '808 publicationgives details regarding this krill oil as derived using those generalsteps identified above.

The published article gives further details of how the processed krilloil alone, at 3000 mgs/daily dose is a product that aids in treatingchronic inflammation and arthritic symptoms. The article describes astudy, which had several objectives: a) to evaluate the effect ofNeptune Krill Oil on C-reactive protein (C-RP) on patients with chronicinflammation; and b) to evaluate the effectiveness of the Neptune KrillOil on arthritic symptoms. The method used a randomized, double blind,placebo controlled study protocol. Ninety patients were recruited witheither a confirmed diagnosis of cardiovascular disease and/or rheumatoidarthritis and/or osteoarthritis and with increased levels of CRP (>1.0mg/dl) upon three consecutive weekly blood analysis prior to initiationof oral treatment with krill oil. It is important to note that C-RP is awell known biomarker for risk of cardiovascular disease, therefore inthis trial, since patients with known cardiovascular disease states wenot excluded from the trial the protocol appears to have evaluated theeffects of krill oil on this cardiovascular risk factor while evaluatingthe effects of krill oil supplementation on the pain and discomfortassociated with OA and RH. Group A received the Neptune Krill Oil (300mg daily) and group B received a placebo. C-RP and Western Ontario andMcMaster Universities (WOMAC) osteoarthritis scores were measured atbaseline and days 7, 14 and 30. After seven days of treatment, theNeptune Krill Oil reduced CRP by 19.3% compared to an increase by 15.7%observed in the placebo group (p=0.049). After 14 and 30 days oftreatment, the Neptune Krill Oil further decreased CRP by 29.7% and30.9% respectively (p<0.001). The CRP levels of the placebo groupincreased to 32.1% after 14 days and then decreased to 25.1% at day 30.The between group difference was statistically significant; p=0.004 atday 14 and p=0.008 at day 30. The application of the processed NeptuneKrill Oil showed a significant reduction in all three WOMAC scores.After seven days of treatment, the Neptune Krill Oil reduced pain scoresby 28.9% (p=0.050), reduced stiffness by 20.3% (p=0.001) and reducedfunctional impairment by 22.8% (p=0.008). The results of that studyindicate that the Neptune Krill Oil at a daily dose of about 300 mgsignificantly inhibits inflammation, reduces arthritic symptoms within ashort treatment period of 7 and 14 days and may be effective in reducingthe risk of cardiovascular disease by reduction of C-RP in the patientpopulation employed.

SUMMARY OF THE INVENTION

In accordance with a non-limiting example, even more beneficial andsynergistic effects for alleviating joint pain and symptoms ofosteoarthritis and/or rheumatoid arthritis have been found when krilloil and/or marine oil are used in combination with other activeconstituents.

In accordance with a non-limiting example, the method treats andalleviates symptoms of osteoarthritis and/or rheumatoid arthritis in apatient by administering a therapeutic amount of a composition includinga krill oil in combination with astaxanthin and low molecular weightpolymers of hyaluronic acid or sodium hyaluronate (hyaluronan) in anoral dosage form. The krill oil in one example is derived from Euphasiaspp., comprising Eicosapentaenoic (EPA) and Docosahexaenoic (DHA) fattyacids in the form of triacylglycerides and phospholipids, although notless than 1% EPA and 5% DHA has been found advantageous. In anotherexample, the krill oil includes at least 15% EPA and 9% DHA, of whichnot less than 45% are in the form of phospholipids. The composition canbe delivered advantageously for therapeutic results with 1-4000 mg ofkrill oil delivered per daily dose. In another example, 0.1-50 mgastaxanthin are supplemented to the krill oil per daily dose.

The astaxanthin is preferably derived from Haematococcus pluvialisalgae, Pfaffia, krill, or by synthetic routes, in the free dial,monoester or diester form. These polymers of hyaluronic acid or sodiumhyaluronate (hyaluronan) can be derived from microbial fermentation oranimal tissue. About 1-500 mg of hyaluronan can be delivered per dailydose. The hyaluronan is micro- or nano-dispersed within the compositionin one preferred example. In a preferred example, the hyaluronic acid isderived from a biofermenation process and has surprisingly a relativelylow molecular weight between 0.5 and 100 kilodaltons (kDa). In anotherexample, the polymers of hyaluronic acid or sodium hyaluronate(hyaluronan) are derived from animal tissue and have molecular weightsexceeding 100 kDa. Such high molecular weight hyaluronans are typicallyderived from rooster combs and are reportedly mildly anti-inflammatorythough this fact is somewhat controversial. The literature clearlyindicates that as the molecular weight of hyaluronic acid and its saltsincreases, its immunogenicity drops dramatically. In addition, thescientific literature clearly points out that low molecular weighthyaluronic acids fragments are unexpectedly highly pro-inflammatory withrespect to the innate immune system and would thus not be expected touseful in the treatment of inflammatory disease states and in particularjoint pain associated with OA and/or RH.

The composition may also include a natural or synthetic cyclooxygenase-1or -2 inhibitor comprising aspirin, acetaminophen, steroids, prednisone,or NSAIDs. The composition may also include a gamma-linoleic acid richoil comprising Borage (Borago officinalis L.) or Safflower (Carthamustinctorius L.).

The composition may also include an n−3 (omega-3) fatty acid rich oilderived from fish oil (EPA and DHA), algae oil (EPA and DHA), flax seedoil (ALA), perrila seed oil (ALA) or chin seed oil (ALA) and the n−3fatty acid comprises, either alone or in combination, alpha-linolenic,stearidonic, eicosapentaenoic or docosapentaenoic acid. Soluble orinsoluble forms of Collagen and elastin such as those derived fromhydrolyzed or un-hydrolyzed eggshell membrane can also be added. Thecomposition may also include anti-inflammatory and/or joint healthpromoting compounds comprising at least one of the preparations of thegreen lipped mussel (Perna canaliculus), Boswellia serrata, turmeric(Curcuma longa), stinging nettle (Urtica dioica), Andrographis, Cat'sclaw (Uncaria tomentosa), bromelain, methylsulfonylmethane (MSM),chondroitin sulfate, glucosamine sulfate, s-adenosyl-methionine,proanthocyanidins, procyanidins or flavonoids, and preparations ofhydrolyzed or un-hydrolyzed eggshell membrane. The composition mayinclude naturally-derived and synthetic antioxidants that are added toretard degradation of polyunsaturated fatty acids and/or the potentanti-oxidant astaxanthin.

Different compositions may use different ingredients in combination withthe krill oil, astaxanthin and hyaluronate and be combined withdifferent ingredients and supplemental compositions for more specificpurposes.

A pharmaceutically acceptable composition comprises a krill oil incombination with astaxanthin and hyaluronate and optionally combinedwith glucosamine sulfate, chondroitin sulfate, collagen,methylsulfonmethane, a gamma-linoleic acid or omega-3 fatty acid richoil or cyclooxygenase inhibitor for the treatment of symptoms related tojoint pain or joint diseases including but not limited to osteoarthritisand rheumatoid arthritis.

In yet another example, a dietary supplement acceptable compositioncomprises a krill oil in combination with astaxanthin and hyaluronateand optionally combined with glucosamine sulfate, chondroitin sulfate,collagen, methylsulfonmethane, a gamma-linoleic acid or omega-3 fattyacid rich oil and a cyclooxygenase inhibitor for the treatment ofsymptoms related to joint pain or joint diseases including but notlimited to osteoarthritis and rheumatoid arthritis.

In yet another example, a medical food acceptable composition comprisesa krill oil in combination with astaxanthin and hyaluronate andoptionally combined with glucosamine sulfate, chondroitin sulfate,collagen, methylsulfonmethane, a gamma-linoleic acid or omega-3 fattyacid rich oil and cyclooxygenase inhibitor for the treatment of symptomsrelated to joint pain or joint diseases including but not limited toosteoarthritis and rheumatoid arthritis.

In still another example, a composition is formulated in a therapeuticamount to treat and alleviate symptoms of osteoarthritis and/orrheumatoid arthritis, wherein the composition includes a krill oil incombination with astaxanthin and polymers of hyaluronic acid or sodiumhyaluronate (hyaluronan) in an oral dosage form. This composition mayinclude other active constituents as explained and identified aboverelative to the method and composition.

A process for extracting astaxanthin from Haematococcus pluvialis algalbiomass is also disclosed and comprises providing a dried form ofHaematococcus pluvialis, extracting astaxanthin using one of at least apolar and non polar solvent to obtain a slurry, filtering the slurry toseparate the cells from the extract, and obtaining an astaxanthinoleoresin concentrate by removing solvent from the extract. Thisastaxanthin oleoresin concentrate is administered to a human patient inan oral dosage form containing 1 to 50 mg of astaxanthin oleoresincomplex to treat and alleviate symptoms of osteoarthritis and/orrheumatoid arthritis in one example.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, features and advantages of the present invention willbecome apparent from the detailed description of the invention whichfollows, when considered in light of the accompanying drawings in which:

FIG. 1 is a view showing a chemical structure of astaxanthin that can beused in accordance with a non-limiting example.

FIG. 2 shows the MMP3 concentration in osteoarthritis (OA) patientsadministered with an astaxanthin oleoresin complex in accordance with anon-limiting example.

FIG. 3 shows MMP3 results on OA patients administered with placebo.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

The composition includes EPA and DHA functionalized as marinephospholipids and acyltriglycerides derived from krill and esterifiedastaxanthin, and in one non-limiting example, low molecular weightpolymers of hyaluronic acid or sodium hyaluronate (hyaluronan) in anoral dosage form. Some of these components are explained in thefollowing chart:

Components PHOSPHOLIPIDS Percentage (%) PC, PE, PI, PS, SM, CL >40OMEGA-3 (functionalized on PL) >30 Eicosapentaenoid Acid (EPA)* >17 (15%in one example and 10% in another) Docosahexaenoid Acid (DHA)+ >11 (9%in one example and 5% in another) ANTIOXIDANTS (mg/100 g) Astaxanthin,Vitamin A, Vitamin E  >1.25 *>55% of PL-EPA/Total EPA +>55% ofPL-DHA/Total DHA These amounts can vary depending on application andpersons.

As noted before, surprisingly the composition includes apro-inflammatory low molecular weight Hyaluronic Acid (LMWtHA). Naturalhigh molecular weight hyaluronic acid is the major hydrodynamiccomponent of synovial fluid and importantly is known to beimmuno-neutral to the innate immune system. It is nature's bone jointshock absorbent and lubricant. It has been found that there is excellentoral bioavailability of LMWtHA fragments specifically to connectivetissue, which maximizes interaction with target synovial fluid producingcells. Therefore in a preferred composition containing krill oil,astaxanthin and LMWtHA, two anti-inflammatory components are thuscombined with one highly inflammatory component. The scientificliterature indicates that LMWtHA fragments exhibit potentpro-inflammatory behavior. It therefore remains unclear why apro-inflammatory component would elicit a favorable overall response ininflamed joint tissues. It is believed that such pro-inflammatory LMWtHAfragments may promote site repair by simulation of the innate immunesystem repair mechanism and by simulating production of non-immunogenichigh molecular weight HA bringing the joint back to homeostasis, howeverthese are theories. A great deal of work by leading immunologists isstill attempting to unravel all the aspects of the complicated singlingprocesses associated with the innate immune system. Studies using largeanimal models of osteoarthritis have shown that mild immunogenicHyaluronic Acids with molecular weights within the range of 0.5-1.0×10⁶Da (Dalton) were generally more effective in reducing indices ofsynovial inflammation and restoring the rheological properties of SF(visco-induction) than non-immunogenic HA's with molecular weights>2.3×10⁶ Da.

Astaxanthin is a required component of the instant invention. Relatedscientific literature indicates that in a lipopolysaccharide inducedinflammatory rat model, astaxanthin at just 1 mg/kg in vitro and invivo: (1) down regulates TNF-alpha production by 75%; (2) down regulatesprostaglandin E-2 production (PGE-2) by 75%; (3) inhibits nitric oxidesynthase (NOS) expression of nitric oxide by 58%; and (4) these effectson inflammatory markers were nearly as effective as prednisolone in thismodel. FIG. 1 shows an example of the astaxanthin as astaxanthin 3S, 3′S(3,3′-dihydroxy-4,4′-diketo-β-carotene). Such information suggests butdoes not prove that astaxanthin may be an effective standalone productfor the reduction of OA and/or RH pain or other symptomology associatedwith OA and/or RH.

In induced uveitis, astaxanthin also showed dose dependant ocularanti-inflammatory activity by suppression of NO, PGE-2 and TNF-Alpha bydirectly blocking NO synthase activity. Astaxanthin is also known toreduce C-Reactive Protein (C-RP) blood levels in vivo. For example, inhuman subjects with high risk levels of C-RP three months of astaxanthintreatment resulted in 43% of patients serum C-RP levels to drop belowthe risk level. This may explain why C-RP levels dropped significantlyin the Deutsch study. Astaxanthin is so powerful that it has been shownto negate the pro-oxidant activity of Vioxx, a COX-2 inhibitor belongingto the NSAIDS drug class which is known to cause cellular membrane lipidper-oxidation leading to heart attack and stroke. For this reason Vioxxwas removed from the US market. Astaxanthin is absorbed in vitro by lensepithelial cells where it suppresses UVB induced lipid per-oxidativemediated cell damage at umol/L concentrations. In human trialsastaxanthin at 4 mgs/day prevented post exercise joint fatigue followingstrenuous knee exercise when compared to untreated subjects. Theseresults have been shown in:

1) Lee et al., Molecules and Cells, 16(1):97-105; 2003;

2) Ohgami et al., Investigative Ophthalmology and Visual Science44(6):2694-2701, 2003;

3) Spiller et al., J. of the Amer. College of Nutrition, 21(5): October2002; and

4) Fry et al., Univ. of Memphis Human Performance Laboratories, 2001 and2004, Reports 1 & 2.

A preferred composition in one embodiment includes 300 mg of krill oil,45 mg of low molecular weight HA, and 2 mg astaxanthin.

Astaxanthin has potent singlet oxygen quenching activity. Astaxanthintypically does not exhibit pro-oxidant activity unlike β-carotene,lutein, zeaxanthin and Vitamins A and E. Astaxanthin in some studies hasbeen found to be about 50 times more powerful than Vitamin E, 11 timesmore powerful than β-carotene and three times more powerful than luteinin quenching of singlet oxygen. Astaxanthin is also well known for itsability to quench free radicals. Comparative studies have foundastaxanthin to be 65 times more powerful than Vitamin C, 54 times morepowerful than β-carotene, 47 times more powerful than lutein, and 14times more powerful than Vitamin E in free radical quenching ability.

U.S. Pat. No. 5,527,533 (the Tso patent), the disclosure which is herebyincorporated by reference in its entirety, discloses the benefits ofastaxanthin for retarding and ameliorating central nervous system andeye damage. Astaxanthin crosses the blood-brain-retina barrier and thiscan be measured by direct measurement of retinal astaxanthinconcentrations. Thus, Tso demonstrated protection from photon induceddamage of photo-receptors, ganglion and neuronal cell damage.

Studies have shown that HA binds to the surface of dendritic cells(“DC's”) and stimulated T-cells. Blockade of the CD44-HA interactionleads to impaired T-Cell activation both in vitro and in vivo. Studieshave shown that in cancer cell lines, LMWtHA fragments specificallyinduce nitric oxide synthase in dendritic cells. In DC's, NO expressioncaused dendritic cell apoptosis (cell death). DC's are essential T-cellactivators which function by presenting antigens to T-cells, thusapoptosis of DC's may short circuit the adaptive immune system response.This effect was clearly CD44 dependent because pretreatment of DC's withanti-CD44 monoclonal antibodies blocked the NO mediated induction of DCapoptosis. It appears that low molecular weight HA fragments interruptthe normal course of the well known T-cell mediated adaptive immunesystem response. CD44 is a glycoprotein responsible in part forlymphocyte activation (also known as T-cell activation) and is known tospecifically bind to HA. On the other hand as previously discussed lowmolecular weight HA fragments appear to up-regulate the innate immuneresponse, particularly in chronic inflammatory conditions where theinnate immune system may in some way be compromised.

Support for such teachings can be found in:

1) Mummert et al., J. of Immunol. 169, 4322-4331;

2) Termeer et al., Trends in Immunology, Vol. 24, March 2003;

3) Yang et al., Cancer Res. 62, 2583-2591; and

4) KcKee et al., J. Biol. Chem. 272, 8013-8018.

Additional information can be found in the following references: GhoshP. Guidolin D. Semin Arthritis Rheum., 2002 August; 32(1):10-37; and P.Rooney, M. Wang, P. Kumar and S. Kumar, Journal of Cell Science 105,213-218 (1993).

As noted before, krill oil is typically produced from Antarctic krill(euphausia superba), which is a zooplankton (base of food chain). It isone of the most abundant marine biomass of about 500 million tonsaccording to some estimates. Antarctic krill breeds in the pureuncontaminated deep sea waters. It is a non-exploited marine biomass andthe catch per year is less than or equal to about 0.02% according tosome estimates.

It is believed that Krill oil based phospholipid bound EPA and DHAuptake into cellular membranes is far more efficient thantriacylglyercide bound EPA and DHA since liver conversion oftriacylglycerides is itself inefficient and because phospholipid boundEPA and DHA can be transported into the blood stream via the lympathicsystem, thus, avoiding liver breakdown. In addition, krill oilconsumption does not produce the burp-back observed with fish oil basedproducts. Because of this burp-back feature of fish oils, it has beenfound that approximately 50% of all consumers who try fish oil never buyit again.

Astaxanthin has an excellent safety record. A conducted study obtainedthe results as follows:

Oral LD 50: 600 mg/kg (rats);

NOAEL: 465 mg/kg (rats); or

Serum Pharmacokinetics: Stewart et al. 2008

1) T_(1/2): 16 hours;

2) T_(max): 8 hours;

3) C_(max): 65 μg/L.

At eight weeks of supplementation at 6 mg per day, there was no negativeeffect in healthy adults. Spiller et al. 2003.

In accordance with one non-limiting example, astaxanthin has three primesources. 3 mg astaxanthin per 240 g serving of non-farmed raised salmonor a 1% to 12% astaxanthin oleoresin or 1.5-2.5% beadlet derived frommicroalgae. Further verification is reflected in Lee et al., Moleculesand Cells 16(1): 97-105, 2003; Ohgami et al., InvestigativeOphthalmology and Visual Science 44(6): 2694-2701, 2003; Spiller et al.,J. of the American College of Nutrition 21(5): October 2002; and Fry etal., University of Memphis, Human Performance Laboratories, 2001 and2004, Reports 1 and 2.

Although many beneficial and synergistic effects are now being reportedherein have been observed when krill oil is used in combination withother active ingredients, and more particularly, krill oil incombination with astaxanthin and polymers of hyaluronic acid or sodiumhyaluronate in preferably an oral dosage form for the control of jointpain range of motion and stiffness, it should be understood thatdifferent proportions of ingredients and percentages in compositions canbe used depending on end use applications and other environmental andphysiological factors when treating a patient.

In accordance with a non-limiting example, the method treats andalleviates symptoms of osteoarthritis and/or rheumatoid arthritis in apatient by administering a therapeutic amount of a composition includinga krill oil in combination with astaxanthin and polymers of hyaluronicacid or sodium hyaluronate (hyaluronan) in an oral dosage form. Thekrill oil in one example is derived from Euphasia spp., comprisingEicosapentaenoic (EPA) and Docosahexaenoic (DHA) fatty acids in the formof triacylglycerides and phospholipids, although not less than 1% EPAand 5% DHA has been found advantageous. In another example, the krilloil includes at least 15% EPA and 9% DHA, of which not less than 45% arein the form of phospholipids. The composition can be deliveredadvantageously for therapeutic results with 1-4000 mg of krill oildelivered per daily dose. In another example, 0.1-50 mg astaxanthin aresupplemented to the krill oil per daily dose.

The astaxanthin is preferably derived from Haematococcus pluvialisalgae, Pfaffia, krill, or by synthetic routes, in the free dial,monoester or diester form at a daily dose of 0.5-8 mg. The polymers ofhyaluronic acid or sodium hyaluronate (hyaluronan) can be derived frommicrobial fermentation or animal tissue. About 1-500 mg of hyaluronancan be delivered per daily dose and preferably between 10 and 70mgs/dose. The hyaluronan is micro- or nano-dispersed within thecomposition in one preferred example. In another example, the hyaluronicacid is derived from a bio-fermentation process and has a molecularweight between 0.5 and 100 kilodaltons (kDa). In another example, thepolymers of hyaluronic acid or sodium hyaluronate (hyaluronan) arederived from microbial fermentation or animal tissue and have molecularweights exceeding 100 KDa and preferably up to 10⁶ KDa.

The composition may also include a natural or synthetic cyclooxygenase-1or -2 inhibitor comprising for example aspirin, acetaminophen, steroids,prednisone, or NSAIDs. The composition may also include a gamma-linoleicacid rich oil comprising Borage (Borago officinalis L.) or Safflower(Carthamus tinctorius L.), which delivers a metabolic precursor to PGE₁synthesis.

The composition may also include an n−3 (omega-3) fatty acid rich oilderived from fish oil, algae oil, flax seed oil, or chia seed oil andthe n−3 fatty acid comprises alpha-linolenic, stearidonic,eicosapentaenoic or docosapentaenoic acid. Hydrolyzed or unhydrolyzedcollagen and elastin derived from eggshell membranes can also beadvantageously added. The composition may also include anti-inflammatoryand/or natural joint health promoting compounds comprising at least oneof preparations of green lipped mussel (Perna canaliculus), Boswelliaserrata, turmeric (Curcuma longa), stinging nettle (Urtica dioica),Andrographis, Cat's claw (Uncaria tomentosa), bromelain,methylsulfonylmethane (MSM), chondroitin sulfate, glucosamine sulfate,s-adenosyl-methionine, proanthocyanidins, procyanidins or flavonoids.The composition may include naturally-derived and synthetic antioxidantsthat are added to retard degradation of fatty acids and astaxanthin.

Different compositions may use different ingredients in combination withthe krill oil, astaxanthin and hyaluronate and be combined withdifferent ingredients and supplemental compositions for more specificpurposes.

A pharmaceutically acceptable composition comprises a krill oil incombination with astaxanthin and hyaluronate optionally combined withone or more ingredients including but not limited to glucosaminesulfate, chondroitin sulfate, collagen, methylsulfonmethane, agamma-linoleic acid or omega-3 fatty acid rich oil a cyclooxygenaseinhibitor or a lipogenase inhibitor for the treatment of symptomsrelated to joint diseases including but not limited to osteoarthritisand rheumatoid arthritis.

In yet another example, a dietary supplement acceptable compositioncomprises a krill oil in combination with astaxanthin and hyaluronateoptionally combined one or more ingredients, including but not limitedto, glucosamine sulfate, chondroitin sulfate, collagen,methylsulfonmethane, a gamma-linoleic acid or omega-3 fatty acid richoil a cyclooxygenase inhibitor or a lipoxygenase inhibitor for thetreatment of symptoms related to joint diseases including but notlimited to osteoarthritis and rheumatoid arthritis.

In yet another example, a medical food acceptable composition comprisesa krill oil in combination with astaxanthin and hyaluronate andoptionally combined with one or more ingredients including glucosaminesulfate, chondroitin sulfate, collagen, methylsulfonmethane, agamma-linoleic acid or omega-3 fatty acid rich oil, a cyclooxygenaseinhibitor or a lipoxygenase inhibitor for the treatment of symptomsrelated to joint diseases including but not limited to osteoarthritisand rheumatoid arthritis.

In still another example, a composition is formulated in a therapeuticamount to treat and alleviate symptoms of osteoarthritis and/orrheumatoid arthritis, wherein the composition includes a krill oil incombination with astaxanthin and polymers of hyaluronic acid or sodiumhyaluronate (hyaluronan) in an oral dosage form. This compositionincludes other active constituents as explained and identified aboverelative to the method and composition.

A process for extracting astaxanthin from Haematococcus pluvialis algalbiomass is also disclosed and comprises providing a dried form ofHaematococcus pluvialis, extracting astaxanthin using one of at least apolar and non polar solvent to obtain a slurry, filtering the slurry toseparate the cells from the extract, and obtaining an astaxanthinoleoresin concentrate by removing solvent from the extract. Thisastaxanthin oleoresin concentrate is administered to a human patient inan oral dosage form containing 1 to 50 mg of astaxanthin oleoresincomplex to treat and alleviate symptoms of osteoarthritis and/orrheumatoid arthritis in one example.

Other details of the type of CO2 extraction and processing technology(as supercritical CO2 extraction) and peroxidation blocker technologythat can be used are disclosed in commonly assigned U.S. PatentPublication Nos. 2009/0181127; 2009/0181114; and 2009/0258081, thedisclosures which are hereby incorporated by reference in theirentirety.

There now follows further details of an astaxanthin extract that can beformed from haematococcus pluvialis and an associated method forpreparation thereof. As described below, this astaxanthin extract andmethod advantageously can be used alone in an oral dosage form to treatand alleviate symptoms of osteoarthritis and/or rheumatoid arthritis. Itis also applied in combination with krill oil and LMWtHA as describedabove.

In this example, astaxanthin oleoresin complex is derived from algaHaematococcus pluvialis, wherein the astaxanthin oleoresin complex isuseful for relieving pain in Osteoarthritic patients.

It should be understood that application of astaxanthin from the algaeHaematococcus pluvialis for the improvement of muscle function, muscledisorders, muscle diseases and carpal tunnel syndrome are reported. Inthese many studies, astaxanthin oleoresin extracted through supercritical CO2 extraction was used. However, no studies or clinical trialswere carried out using astaxanthin oleoresin specifically foralleviating symptoms of Osteoarthritis.

Osteoarthritis (OA) is the most prevalent form of arthritis.Osteoarthritis is a disease in which the cartilage that acts as acushion between the bones in joints begins to wear away causing bone onbone joint swelling and joint pain. It is characterized by degenerationof articular cartilage along with peri-articular bone response. Itaffects both sexes, mainly in the fourth and fifth decades of life. Theknee joint is most commonly affected joint. At present the management isby pharmacological and non-pharmacological therapy. Corrective surgicaltherapy and or joint replacement therapy in some cases may not bepossible.

Traditional treatments for Osteoarthritis involve the use of analgesics,non-steroidal anti-inflammatory drugs (NSAIDs) or cyclooxygenase-2specific (COX-2) NSAIDs alone or in combination. Advances in recombinantprotein synthesis also provide relief from the symptoms of OA and RH.Steroid or high molecular weight hyaluronic acid injections have alsobeen used with some success however these therapies have well knowndeleterious side effects.

Many of these treatments alone have shown limited effectiveness inclinical trials. To avoid the cardiac risks and gastrointestinal issuesassociated with traditional OA treatments (particularly with long termuse), many patients have turned to complimentary and alternativemedicines (CAMs) such as dietary supplements. Glucosamine andchondroitin alone or in combination, are widely marketed as dietarysupplements to treat joint pain due to OA. Two major clinical trials onglucosamine and chondroitin (The GAIT Study) failed to show anysignificant improvement in WOMAC score over placebo except in thehighest quartile of patients studied. Because of their limitedeffectiveness, the search for additional CAMs to treat OA continues (seefor example Ruff et al., Eggshell membrane in the treatment of pain andstiffness from Osteoarthritis of the knee: a randomized, multicenter,double-blind, placebo-controlled clinical study, Clin. Rheumatol (2009)28:907-914).

In accordance with a non-limiting example, a composition comprisesastaxanthin oleoresin complex derived form Haematococcus pluvialis algalbiomass as a single component treatment to relieve the symptoms of OA. Aprocess for extraction of the astaxanthin oleoresin complex from algaHaematococcus pluvialis is also provided. A use of the astaxanthinoleoresin complex is provided wherein the astaxanthin oleoresin complexis useful for relieving pain in Osteoarthritis patients whenadministered in an effective amount. The astaxanthin oleoresin complexas disclosed is helpful in improving the quality of life, functionalityand by administering required amount of Astaxanthin oleoresin complex.

The astaxanthin oleoresin complex as disclosed is derived by extractionof the Haematococcus pluvialis algal biomass by solvent extraction usingpolar/non-polar solvents in a non-limiting example. The Haematococcuspluvialis biomass is obtained from cultivating the alga in open ponds,harvesting astaxanthin rich biomass, drying the harvested biomass toremove the moisture and cracking the dried biomass for extraction ofastaxanthin complex from the said algal biomass.

The subject matter relates to long term management of the patients withOsteoarthritis in terms of a) relief of pain and b) improved functionsof daily life using astaxanthin oleoresin complex as disclosed.

In one of embodiment, there is provided a composition comprisingastaxanthin oleoresin complex. In another embodiment, there is provideda composition comprising the astaxanthin oleoresin complex derived fromHaematococcus pluvialis algal biomass. In another embodiment, there isprovided a process for extraction of astaxanthin from Haematococcuspluvialis algal biomass, wherein the process comprises providing driedform of Haematococcus pluvialis, extracting astaxanthin using polarand/or non-polar solvents to obtain a slurry; filtering the slurry toseparate the cells from extract; and obtaining astaxanthin oleoresinconcentrate by removing solvent from the extract. In another embodiment,there is provided Haematococcus pluvialis alga, wherein the alga iscultivated in open ponds or in closed photobioreactors. One embodimentprovides the process for extraction of astaxanthin Haematococcuspluvialis algal biomass, wherein the Haematococcus alga accumulated withastaxanthin is harvested to concentrate the biomass. In anotherembodiment, there is provided a process for extraction of astaxanthinHaematococcus pluvialis algal biomass, wherein red cysts of theHaematococcus pluvialis are dried to remove the moisture. In anotherembodiment, there is provided a process for extraction of astaxanthinHaematococcus pluvialis algal biomass, wherein red cysts of theHaematococcus pluvialis are dried to remove the moisture and the driedred cysts are cracked for optimal extraction of the astaxanthin. Amethod improves the quality of life and functionality of Osteoarthritispatients by administering required amounts of astaxanthin oleoresincomplex in oral dosage form.

In one embodiment, there is provided a method of improving the qualityof life and functionality of Osteoarthritis patients by administeringastaxanthin oleoresin complex in oral dosage forms containing 1-50 mgastaxanthin oleoresin complex. In another embodiment, there is provideda method of improving the quality of life and functionality ofOsteoarthritis patients by administering astaxanthin oleoresin complexin oral dosage forms containing 1-30 mg astaxanthin oleoresin complex.In another embodiment, there is provided a method of improving thequality of life and functionality of Osteoarthritis patients byadministering astaxanthin oleoresin complex in oral dosage formscontaining 1-20 mg astaxanthin oleoresin complex. In another embodiment,there is provided a method of improving the quality of life andfunctionality of Osteoarthritis patients by administering astaxanthinoleoresin complex in oral dosage forms containing 10-25 mg astaxanthinoleoresin complex. In one embodiment, there is provided a method ofimproving the quality of life and functionality of Osteoarthritispatients by administering astaxanthin oleoresin complex in oral dosageforms containing 15 mg astaxanthin oleoresin complex. In anotherembodiment, there is provided a method of improving the quality of lifeand functionality of Osteoarthritis patients by administeringastaxanthin oleoresin complex in oral dosage forms containing 3-8 mg,preferably 4 mg, most preferably 5 mg astaxanthin oleoresin complex. Inanother embodiment, there is provided a method of improving the qualityof life and functionality of Osteoarthritis patients by administeringastaxanthin oleoresin complex in oral dosage forms containingastaxanthin oleoresin complex, wherein the Osteoarthritis assessment inOsteoarthritis patients is based on Western Ontario and McMastersUniversities (WOMAC) Osteoarthritis Index.

In one of the embodiment, there is provided a dietary supplementformulation comprising astaxanthin oleoresin complex, either alone or inconjunction with surfactants to improve the absorption of astaxanthinand therefore the bio-availability of astaxanthin wherein theformulation is useful for improving the quality of life, functionalityand relief of pain in Osteoarthritis, wherein the formulation comprises1-50 mg astaxanthin oleoresin complex. In another embodiment, there isprovided a pharmaceutical formulation comprising astaxanthin oleoresincomplex, either alone or in conjunction with surfactants to improve theabsorption of astaxanthin and therefore the bio-availability ofastaxanthin, wherein the formulation is useful for improving the qualityof life, functionality and relief of pain in Osteoarthritis, wherein theformulation comprises 1-50 mg astaxanthin oleoresin complex. In yetanother embodiment, there is provided a food product comprisingastaxanthin for improving the quality of life, functionality and reliefof pain in Osteoarthritis, wherein the formulation comprises 1-50 mgastaxanthin oleoresin complex either alone or in conjunction withsurfactants to improve the absorption of astaxanthin and therefore thebio-availability of astaxanthin. In yet another embodiment, there isprovided food and/or beverage product comprising astaxanthin oleoresincomplex either alone or in conjunction with surfactants to improve theabsorption of astaxanthin and therefore the bio-availability ofastaxanthin for improving quality of life, functionality and relief ofpain in Osteoarthritis, wherein the formulation comprises 1-50 mgastaxanthin oleoresin complex. One embodiment provides the astaxanthinformulations as disclosed comprising 2 to 20 mg of astaxanthin.

Cultivation of Haematococcus pluvialis Alga

Haematococcus is a uni-cellular green microalga which has the capacityto concentrate in its cells the xanthophyll/carotenoid pigmentastaxanthin under appropriate growth conditions. Astaxanthin has acharacteristic red color. Haematococcus alga can concentrate astaxanthinup to 1.0% to 2% of its body weight in open pond cultivation or up to 5%in photobioreactors. The alga is grown in open ponds containing waterenriched with nutrients of nitrates, phosphate, magnesium, CO2, mineralsetc., for the algal growth. The cultivation of the alga is not acontinuous process and is carried out under a batch process of 7-10days.

The Astaxanthin rich alga is separated/harvested using mechanicalmethods/separator machines from the pond culture medium and the filtratefrom the pond is cleaned and recycled. The harvested algal slurry iswashed using fresh water and concentrated. Thereafter the slurry in wetform along with antioxidants is dried through a spray drier. The powderis stored under normal warehouse condition and or in cold storage andthen cracked and stored under nitrogen or vacuum packed. An example isgiven in Indian Patent No. 201526 (Swati S Thomas, Kumaravel S and JeejiBai N-2003-A cyclic process for the production of astaxanthin enrichedbiomass from Haematococcus algae) on “A cyclic process for theproduction of astaxanthin enriched biomass from Haematococcus algae.”

Extraction Process for the Preparation of Astaxanthin Oleoresin Complex

The dried form of Haematococcus pluvialis alga is used as the rawmaterial for the extraction process. The algal powder is cracked using acracking mill. The cracked cells are then immersed in a suitablepolar/non-polar solvent and mixed. After giving sufficient time forastaxanthin extraction, the mixture is passed through a filtration unitto separate the cells from the extract. The spent cell with solvent isthen passed through a unit to remove solvent from the cells. The clearfiltrate with astaxanthin is then passed through a distillation unit toremove the solvent. The concentrated astaxanthin is finally passedthrough high vacuum distillation unit to strip the extraction solventresidue below 100 ppm level. All the operations are carried out undervacuum or under nitrogen atmosphere to avoid any degradation.Astaxanthin oleoresin is red in color and the concentration ofastaxanthin will range from 2.5% to 10% depending on the level ofastaxanthin in the algal powder.

Composition of Haematococcus pluvialis Cell Powder & AstaxanthinOleoresin Complex

Astaxanthin (3,3′-dihydroxy-b, b-carotene-4,4′-dione) is widelydistributed in nature and is the principle pigment in crustaceans andSalmonoids, various birds including flamingos and scarlet ibis. Thecarotenoids impart distinctive orange red coloration (Bjørn Bjerkeng,Marianne Føllingb, Stephen Lagockia, Trond Storebakkena, Jan Ollic andNiels Alstedd. Bioavailability of all-Eastaxanthin and Z-isomers ofastaxanthin in rainbow trout (Oncorhynchus mykiss). Aquaculture, (1997).157: 63-82.

Astaxanthin is a keto (oxygenated) carotenoid with the molecular formulaC₄₀H₅₂O₄ and has a molecular weight of 596.86. Astaxanthin exists inseveral stereochemical forms viz 3S, 3′S; 3R, 3′R; 3S, 3′R and 3R, 3′Sdepending on the source (Schiedt, K., Leuenberger, F. J., Vecchi, M.Analysis of astaxanthin as found in wild salmon and syntheticastaxanthin, Helv. Chim. Acta (1981) 64: 449-457). The major naturalsources of astaxanthin include algae Haematococcus, yeast Phaffia andKrill apart from salmon and trout. Among these, Haematococcus is therichest source producing between 1% to 4% astaxanthin of its dry weight(Maher, T. J. Astaxanthin: A versatile carotenoid antioxidant.International Journal of Integrative Medicine (2000) Volume 2 (number4)). In Haematococcus algae astaxanthin exists as 3S, 3′S isomer,whereas in the yeast it is in the form of 3R, 3′R isomer. Astaxanthinexists in Haematococcus cells in the form of monoester and diester,which accounts for more then 90% of total astaxanthin. Other carotenoidssuch as beta-carotene, canthaxanthin and lutein are present in minoramounts (Lee, Y-K., Mang, and D. H. Production of astaxanthin byHaematococcus. In: Chemicals from Microalgae. Ed: Zvi Cohen, Taylor andFrancis, UK (1999). Pp. 173-195). Astaxanthin exists in free form insynthetic astaxanthin and Phaffia yeast. The summary of results is givenTable 1 and 2.

Clinical Trial to Evaluate the Efficacy of Haematococcus pluvialisAstaxanthin Oleoresin Complex in Osteoarthritis Patients

The study has been carried out as a comparative single blind clinicaltrial of astaxanthin oleoresin complex in 60 Osteoarthritis patients ascompared with Placebo control for a period of 12 weeks n=60 (30 A+30 P).The dosage consisted of one softgel containing 15 mg of Astaxanthin oncea day during breakfast for 12 weeks. A total of 70 subjects wererecruited for the study, 35 in each group (Astaxanthin oleoresin complexand placebo-control) of both the sexes. Patients were explained thenature of the study and informed consent was obtained prior to the startof the study. Patient subjects were clinically examined by the PrincipalInvestigator and team. X ray and blood samples were drawn at thecommencement and at the end of study period. The case record forms werefilled by the Principal Investigator and rechecked by the Clinicalresearch associate. Sixty patient subjects completed the study. Ten weredrop outs due to various reasons but not on account of intolerance tothe astaxanthin oleoresin complex or placebo control. The results weretabulated by the expert data entry operators under supervision ofBiometric expert. The results were subjected to Statistical analysis byan independent analyst.

The assessment of Osteoarthritis symptoms were based on Western Ontarioand McMasters Universities (WOMAC) Osteoarthritis Index, VAS scale,Lequesne's functional scale as well as Sleep score as additionalparameters besides radiological investigations. Further the assessmentof Osteoarthritis symptoms based on haematological studies, specificallyMMP3 (Matrix metalloproteinase 3) in clinical parameters sinceOsteoarthritis patients show elevated levels of MMP3 in blood as well asin synovial fluid. The elevated levels cause significant tissue damagethrough cartilage destruction.

Results of Clinical Trial and Discussions

Total Health Assessment Score—The total health assessment onOsteoarthritis patients was carried out on their difficulty to a)Dressing—doing buttons, washing and combing hair b) Arising—stand upstraight from a chair, get in and out of bed, sit cross-legged on floorand get up c) Eating—cut vegetables, lift a full cup/glass to your mouthd) Walking—walk outdoor on flat ground, climb up five steps and e)Hygiene-Take a bath, wash and dry your body, get on and off the toiletf) Reaching—reach and get down a 2 kg object from just above your head,bend down to pick up clothing from the floor g) Grip-open a bottlepreviously opened, turn taps on & off, open door latches h)Activities-work in office/house, run errand to shop, get in and out ofcar/auto. The summary of results is given Table 3.

There were significant reductions in the mean scores of patients takingastaxanthin oleoresin complex at the end of 3 months but not for thePlacebo group. There were no significant differences between astaxanthinand Placebo group at Basal values. There were significant differencesbetween the astaxanthin and placebo group at 3 months.

WOMAC Score—The Western Ontario McMaster (WOMAC) is a validatedinstrument designed specifically for the assessment of lower extremitypain and function in Osteoarthritis (OA) of the knee. The patients wereassessed on their pain, stiffness and difficulty in carrying outday-to-day activities. The pain index was assessed for Activities—a) inwalking on flat surface, going up or down on flat surface, at nightwhile in bed, sitting or lying, standing upright b) Stiffness—afterfirst wakening in morning, after sitting/lying or resting later in theday and c) difficulty in descending stairs, ascending stairs, standingup from a chair, while standing, bending to floor to pick up objects,walking on flat ground, getting in & out of autorickshaw/bus/car, goingshopping, on rising from bed, while lying on bed, while sitting onchair, going on/off toilet, doing heavy domestic duties such as movingheavy boxes/scrubbing floor/lifting shopping bags, doing light domesticduties such as cleaning room/table/cooking/dusting, while sittingcross-legged position, rising from cross-legged position, whilesquatting on floor. The summary of the results are given in Table 4.

There were significant reductions in the mean scores for patients takingAstaxanthin oleoresin complex at the end of 3 months but not for thePlacebo group. There were no significant differences between patientstaking Astaxanthin oleoresin complex and placebo group at basal values.There were significant differences between Astaxanthin and Placebogroups at 3 months.

VAS (Visual Analog Scale) on Pain Parameters—Pain parameters wereassessed in Osteoarthritis patients taking astaxanthin oleoresin and thePlacebo group using VAS. The assessment was carried out in a) Painparameters—pain while using stairs, pain while walking on flat ground,pain while standing upright, pain while sitting or lying down, pain atnight in bed b) Physical functions—going downstairs, going upstairs,sitting, getting up from sitting, standing, bending to floor, walking onflat ground, getting into or out of automobiles, shopping, putting onsocks/stockings, taking off socks/stockings, getting into bed, gettingout of bed, getting into or out of bath tub, getting on or off toiletseat, during heavy household chores, during light household chores,getting into lotus position. The summary of results of Pain parameters(Pain+Physical) scores are given in Table 5.

There were significant reductions in the mean scores at the end of 3months for patients taking Astaxanthin oleoresin complex but not for thePlacebo group. There were no significant differences between Astaxanthinoleoresin complex and Placebo group at Basal values. There weresignificant differences between Astaxanthin oleoresin complex andPlacebo groups at 3 months.

Laquesne's Index—Laquesne's index is the Functional index forOsteoarthritis of the knee. Assessment is carried out on a)Pain/discomfort—during nocturnal bed rest, morning stiffness orregressive pain after rising, after standing for 30 minutes and b)Physical functions—maximum distance walked, activities of daily livinglike able to climb up a standard flight of stairs, able to climb down astandard flight of stairs, able to squat or bend on the knees, able towalk on uneven ground. The Laquesne's index results are given in Table6.

There were significant reductions in mean scores for the patients takingAstaxanthin oleoresin complex at the end of 3 months but not for thePlacebo group. There were no significant differences between astaxanthinoleoresin complex and Placebo groups at Basal values. There weresignificant differences between astaxanthin oleoresin complex andPlacebo groups at 3 months.

Sleep Scale—Sleep is an important element of functioning and well being.Sleep Scale was originally developed in the Medical Outcomes Study (MOS)intended to assess the extent of sleep problems. The Medical OutcomesStudy Sleep Scale includes 12 items assessing sleep disturbance, sleepadequacy, somnolence, quantity of sleep, snoring, and awakening short ofbreath or with a headache. A sleep problems index, grouping items fromeach of the former domains, is also available. This assessment evaluatedthe psychometric properties of MOS-Sleep Scale in Osteoarthritispatients taking Astaxanthin oleoresin complex and Placebo group. Theresults on Sleep scale MOS is given in Table 7.

There were significant reductions in the mean scores for patients takingastaxanthin oleoresin complex at the end of 3 months but not for thePlacebo group. There were no significant differences between astaxanthinoleoresin complex group and Placebo group at Basal values. There weresignificant differences between astaxanthin oleoresin complex group andPlacebo group for most of the variables.

MMP3 (Matrix Metalloproteinase 3) Assay—Assessment of Osteoarthritissymptoms based on haematological studies, specifically MMP3 (Matrixmetalloproteinase 3) were carried out in clinical parameters sinceOsteoarthritis patients show elevated levels of MMP3 in blood as well asin synovial fluid. The elevated levels cause significant tissue damagethrough cartilage destruction. The results of the MMP3 analysis onOsteoarthritis patients before and after 3 months of administering withastaxanthin oleoresin complex are given in FIG. 2. The results of theMMP3 analysis on Osteoarthritis patients before and after 3 months ofadministering with Placebo are given in FIG. 3. MMP3 levels did not showsignificant change but the trend is towards reduction.

In all, 70 subjects were recruited for the study in a randomized manner.The patients were explained the nature of the study as well as active(astaxanthin oleoresin complex softgels containing 15 mg astaxanthin)and placebo treatments. An informed written consent was obtained fromthe subjects prior to the commencement of the study. At the commencementof the study patient subjects were clinically examined and blood sampleswere collected for CBC/ESR & MMP3 study. Specific orthopaedic andradiological examinations were performed. The patient subjects wereassigned placebo and active treatment in a random manner for a period of12 weeks. Patient subjects were advised to continue with their otherroutine treatments, if any. At the end of 4 weeks the subjects werecalled for a second visit in order to refill the samples. The sameprocedure was carried out in third visit and the procedure of the firstvisit was repeated in fourth visit. Results were tabulated by data entryoperators and detailed statistical analysis was performed using thoseresults. At the base level the groups were similar and comparable.

Advantages of the Invention

Total Health Assessment score (Arising, Dressing, Eating, Walking,Hygiene, Grip, Reaching, Daily activities) exhibited significant changesbetween Astaxanthin oleoresin complex and Placebo group (P<0.001).Improvement was seen in all the parameters of daily activities.

WOMAC INDEX exhibited significant differences (P<0.001). This score isunique for the functional abilities in patients with chronic jointdisorders such as Osteoarthritis.

VAS Pain parameters (Pain+Physical) score: There were significantreductions in the mean scores at the end of treatment for patientstaking astaxanthin oleoresin complex but not for Placebo P (<0.001). Itis suggestive of improvement in the pain related aspects ofOsteoarthritis.

Laquesne's index: (Functional Index for OA of knee): There weresignificant reductions in the mean scores at the end of treatment forpatients taking Astaxanthin oleoresin complex but not for Placebo(P<0.05).

Sleep scale from the medical outcomes study: There were significantreductions in the mean scores at the end of treatment for patientstaking astaxanthin oleoresin complex but not for Placebo (P<0.001).

There was significant difference between the average sleep each night(hrs). Patients taking astaxanthin oleoresin complex had higher sleepthan Placebo group (P<0.01).

Improvement in the sleep time clearly indicates efficacy of thetreatment with astaxanthin oleoresin complex. Astaxanthin helps to getbetter sleep as is evident from sleep score. This is due to reduction inpain and other symptoms of the disorder MMP3 did not show significantchange but the trend is towards reduction. Reduction in MMP3 levels aresuggestive of improving cartilage health due to reduction in the processof cartilage destruction in a positive manner although there is neitherdirect proof to this effect nor statistically significant effect in thepresent study. No change in the radiological picture was seen. Nonoteworthy side effect/intolerance was noted during the study period.Astaxanthin oleoresin complex appears to be safe for generalconsumption.

Astaxanthin oleoresin complex extracted through polar solvents fromHaematococcus pluvialis alga may be suitable for the patients in theearly stage of the Osteoarthritis to prevent the progression of thedisorder. It may be useful to the patients with establishedOsteoarthritis to provide symptomatic relief from pain and improvedquality of life. Astaxanthin oleoresin complex improves symptoms likepain as well as quality of physical activities of daily life in asignificant manner. Osteoarthritis is seen to mark its presence at ayounger age in India. It would be appropriate to initiate the treatmentwith Astaxanthin oleoresin complex right from the beginning as soon asthe diagnosis is arrived at. Study with larger sample size at differentcentres is recommended to study the mechanism of action of Astaxanthinoleoresin complex in Osteoarthritis further.

TABLE 1 Carotenoid Profile of Haematococcus Pluvialis Cell Powder andAstaxanthin Oleoresin Complex Astaxanthin Oleoresin complex CarotenoidsCell powder 5% Beta-carotene 0.62 ± 0.01 0.62 ± 0.01 Canthaxanthin 1.21± 0.03 1.20 ± 0.03 Astacene 3.09 ± 0.06 3.09 ± 0.06 Semiastacene 1.35 ±0.03 1.35 ± 0.03 Dicis 1.07 ± 0.02 1.03 ± 0.05 astaxanthin Trans 75.70 ±1.53  75.75 ± 1.51  astaxanthin 9 cis 9.20 ± 0.77 9.19 ± 0.77astaxanthin 13 cis 6.10 ± 0.94 6.08 ± 0.93 astaxanthin Lutein 1.66 ±0.03 1.65 ± 0.03

TABLE 2 Proximate Analysis, Carotenoid Profile and Fatty Acid Profile ofAstaxanthin Oleoresin Complex Astaxanthin oleoresin PARAMETER complex 5%PHYSICAL Appearance Free flow Color Dark red PROXIMATE Protein % 0.95 ±0.03 Carbohydrate % 0.11 ± 0.01 Lipid % 94.89 ± 0.12  Ash % 3.82 ± 0.08Moisture % 0.23 ± 0.02 Carotenoids 5.14 ± 0.04 CAROTENOIDS % Totalcarotenoids 5 Total astaxanthin 4.68 [all-trans-astaxanthin [3.909-cis-astaxanthin 0.47 13-cis-astaxanthin 0.31 15-cis-astaxanthin 0Dicis - astaxanthin] 0.05] Betacarotene 0.03 Canthaxanthin 0.06 Lutein0.08 FATTY ACID PROFILE, Area % C14:0 Myristic acid 0.23 C 15:0Pentadecanoic acid 0.1 C 16:0 Palmitic acid 24.57 C16:1 Palmitoleic acid0.57 C 16:2 Hexadeca dienoic acid 0.45 C 16:3 Hexadecatrienoic acid 0.14C 16:4 Hexadecatetraenoic acid 1.15 C17:0 Heptadecanoic acid 2.14 C 18:0Stearic acid 1.61 C18:1 Oleic acid 38.93 C 18:2 Linoleic acid 17.22 C18:3, n − 6 Gamma linolenic 0.84 acid C 18:3, n − 3 Alpha linolenic 8.14acid C 18:4 Octadeca tetraenoic acid 1.3 C20:2 Eicosadienoic acid 0.81C20:4 Arachidonic acid 0.85 C22:0 Behenic acid 0.5

TABLE 3 Total Health Assessment Score Total Health Assessment ScoreDuration Significance Treatments Basal 1 month 2 months 3 months levelAstaxanthin 18 14.68 13.19 12.13 S, P < 0.001 Placebo 20.25 19.8 19.4819.51 NS, P = 0.4 S = Significant, NS = Not Significant, P = Probability

TABLE 4 WOMAC Score WOMAC Duration Significance Treatments Basal 1 month2 months 3 months level Astaxanthi 36.3 31.87 28.42 26.52 S, P < 0.001Placebo 38.07 36.62 36.59 36.1 NS, P = 0.6 S = Significant, NS = NotSignificant, P = Probability

TABLE 5 VAS Pain Parameters Score Pain Parameters Duration SignificanceTreatments Basal 1 month 2 months 3 months level Astaxanthi 891.94828.71 772.58 748.39 S, P < 0.001 Placebo 945.86 923.28 916.21 915.17NS, P = 0.1 S = Significant, NS = Not Significant, P = Probability

TABLE 6 Laquesne's Index Significance Parameters Astaxanthin Placebolevel 1. During nocturnal bed rest Basal 0.6 +/− 0.7 0.6 +/− 0.7 NS, P =1.0 3 months 0.8 +/− 0.7 0.5 +/− 0.7 S, P = 0.05 2. Morning stiffness orRegressive pain after rising Basal 0.9 +/− 0.6 0.6 +/− 0.7 NS, P = 0.9 3months 0.6 +/− 0.6 0.6 +/− 0.5 NS, P = 0.9 3. After standing for 30Minutes Basal 0.4 +/− 0.5 0.6 +/− 0.7 NS, P = 0.9 3 months 0.3 +/− 0.60.5 +/− 0.7 S, P = 0.05 4 . Maximum distance walked Basal 1.3 +/− 0.71.7 +/− 1.3 NS, P = 0.9 3 months 0.6 +/− 0.5 1.7 +/− 1.3 S, P = 0.001 5.Activities of daily living a) Able to climb up a standard flight ofstairs Basal 0.8 +/− 0.5 0.9 +/− 0.3 NS, P = 0.9 3 months 0.7 +/− 0.51.0 +/− 0.4 S, P = 0.03 b) Able to climb down a standard flight ofstairs Basal 1.3 +/− 0.3 1.6 +/− 0.9 NS, P = 0.9 3 months 0.9 +/− 0.61.6 +/− 0.9 S, P = 0.03 c) Able to squat or bend the Knees Basal 1.3 +/−0.3 1.6 +/− 0.9 NS, P = 0.9 3 months 0.9 +/− 0.6 1.6 +/− 0.9 S, P = 0.03d) Able to walk on uneven Ground Basal 1.3 +/− 0.3 1.6 +/− 0.9 NS, P =0.9 3 months 0.9 +/− 0.6 1.6 +/− 0.9 S , P = 0.03 S = Significant, NS =Not Significant, P = Probability

TABLE 7 Sleep Scale MOS Significance Sleep parameters AstaxanthinPlacebo level  1. Time to fall asleep (min) during the past 4 weeksBasal 2.3 +/− 1.3 2.6 +/− 1.3 NS, P = 0.9 3 months 1.6 +/− 1.1 2.5 +/−1.3 S, P < 0.001  2. Average sleep each night (hours during last 4weeks)  6. +/− 1.3  5. +/− 1.8 S, P < 0.001  3. Feel your sleep was notquiet? Basal  3. +/− 1.9  3. +/− 1.9 NS, P = 0.9 3 months 2.6 +/− 2.13.8 +/− 2.1 S, P = 0.02  4. Get enough sleep to feel rested upon? Basal3.8 +/− 1.9 3.8 +/− 1.9 NS, P = 1.0. 3 months 2.9 +/− 2.1 3.6 +/− 2.1 S,P = 0.03  5. Awaken short of breath or with headache? Basal 5.6 +/− 1.24.6 +/− 2.2 NS, P = 0.6 3 months 5.6 +/− 1.2 4.5 +/− 2.8 NS, P = 0.6  6.Feel drowsy or sleepy during day? Basal  5. +/− 1.8  4. +/− 2.2 NS, P =0.6 3 months 5.7 +/− 1.8 4.5 +/− 2.8 NS, P = 0.6  7. Have troublefalling asleep? Basal 3.6 +/− 2.7 4.6 +/− 2.2 NS, P = 0.3 3 months 4.4+/− 2.1 4.5 +/− 2.8 NS, P = 0.9  8. Awaken during your sleep time andhave trouble in Basal 4.1 +/− 2.9 4.6 +/− 2.2 NS, P = 0.7 falling sleepagain? 3 months 4.9 +/− 2.3 4.5 +/− 2.8 NS, P = 0.7  9. Have troublestaying awake during the day? Basal 4.7 +/− 1.8 4.6 +/− 2.2 NS, P = 0.93 months 5.4 +/− 1.8 4.5 +/− 2.8 S, P = 0.05 10. Snore during yoursleep? Basal 5.5 +/− 1.1 4.4 +/− 1.5 NS, P = 0.2 3 months 5.7 +/− 0.84.8 +/− 1.3 S, P = 0.05 11. Take naps (5 min. or longer during the day?Basal 4.1 +/− 1.5 4.4 +/− 1.5 NS, P = 0.6 3 months 3.7 +/− 1.5 4.8 +/−1.3 S, P = 0.05 12. Get the amount of sleep you needed? Basal 3.2 +/−1.8 4.5 +/− 1.5 NS, P = 0.6 3 months 3.7 +/− 1.8 4.8 +/− 1.3 S, P = 0.05S = Significant, NS = Not Significant, P = Probability

Many modifications and other embodiments of the invention will come tothe mind of one skilled in the art having the benefit of the teachingspresented in the foregoing descriptions and the associated drawings.Therefore, it is understood that the invention is not to be limited tothe specific embodiments disclosed, and that modifications andembodiments are intended to be included within the scope of the appendedclaims.

That which is claimed is:
 1. A process for extracting astaxanthin fromHaematococcus pluvialis algal biomass, comprising: providing a driedform of Haematococcus pluvialis cells; extracting astaxanthin usingsupercritical fluid extraction with one of at least a polar and nonpolar solvent to obtain a slurry; filtering the slurry to separate thecells from the extract; and obtaining an astaxanthin oleoresinconcentrate by filtering and distilling to remove solvent from theextract, wherein the steps of extracting, filtering, and distillingoccur under an oxygen free nitrogen atmosphere.
 2. A process forproducing astaxanthin oleoresin, comprising: obtaining an algal powderof dried Haematococcus pluvialis cells; cracking the algal powder toform cracked cells; extracting astaxanthin from the cracked cells usingsupercritical fluid extraction with one of at least a polar andnon-polar solvent to obtain a slurry; filtering the slurry to separatethe cells from the extract; and distilling the filtrate to remove thesolvent and produce an astaxanthin oleoresin, wherein the steps ofextracting, filtering, and distilling occur under an oxygen freenitrogen atomsphere.
 3. The process according to claim 2, wherein thestep of distilling comprises passing the astaxanthin oleoresin through ahigh vacuum distillation unit to strip the extraction solvent residue tobelow 100 ppm.
 4. The process according to claim 2, wherein the step ofcracking comprises cracking the algal powder using a cracking mill. 5.The process according to claim 2, wherein the step of obtaining an algalpowder comprises processing the algal powder under vacuum.
 6. Theprocess according to claim 2, wherein the concentration of astaxanthinoleoresin comprises 2.5 to 10 percent.